© 2011 Nature America, Inc. All rights reserved. purposes ligand/receptor interactions). non determining for able invalu be would techniques Such (RFP). protein fluorescent red with labeled glia and GFP with labeled tissues—neurons of types one might want to differentially label and manipulate two different then examine the effects on the other population of cells. Likewise, able to express an effector in a fraction of a cellular population, and by GAL4. defined In often complexorganisms, desir is it cellular cells of population single the in expressed be only can effectors allows for a rich investigations.diversity experimental of (GAL4, components GAL4 three the of a Repressible Cell Marker (MARCM) technique for further effector refinement, including the Mosaic Analysis with GAL4,expressed with GAL4 of suppressor natural GAL80, a RNAi constructs). Furthermore, GAL4 activity can be inhibited by or neurotoxins (e.g., molecules inhibitory to Ras) activated (e.g., from cell markers (e.g., membrane range lines Effector lines. effector of thousands with tion in patterns expression developmental and tissue for have lines characterized GAL4 been geneX and GAL4 when Only reporter transgene ( sequence ( activation upstream an flies in manipulations experimental revolutionized has it system, and expression binary repressible a such is system GAL4 yeast The element. exogenous an by inhibited specifically be can itself factor transcription the if afforded is control of layer additional factor. An transcription uses a promoter that is specifically activated only by the introduced ing an exogenous transcription factor, whereas the other transgene In such a strategy, one transgene contains a specific organisms. promoter multicellular driv in tissues and cells of function or ment of an effector transgene for the purpose of investigating the Binarydevelop­ expression is a powerful strategy for regulating the I mosaic analysis. expression patterns that precisely limit which tissues will be experimentally manipulated and how to use the Q system to perform effector transgenes in tissues of interest, how to use the Q system in conjunction with the G for performing a variety of Q system experiments purpose, we recently developed the Q system, a second repressible binary expression system. We describe here the basic steps However, complex biological systems often require distinct transgenic ofmanipulations different cell populations. For this In Published online 7 July 2011; doi:10.1038/nprot.2011.347 Hughes Medical Institute, Stanford University, Stanford, California, USA. Correspondence should be addressed to C.J.P. ([email protected]). 1 Christopher J Potter Using the Q system in work. The Q system uses genes from the the from genes uses system Q The work. The The Solomon H. Snyder Neuroscience,of Department The Johns Hopkins University Medicine,School of Baltimore, Maryland, USA. NTRO We have recently characterized the Q system for these and other limitations. its has system GAL4 the Nonetheless, Drosophila expressed in the GAL4 expression pattern. Thousands of of Thousands pattern. expression GAL4 the in expressed D UCT 4 ; the protocol described here is based on this previous previous this on based is here described protocol the ; I , , the G ON T he he protocol described here can be adapted to a wide range of experimental designs. UAS-geneX AL 1, UAS-geneX 4/ 2 Drosophila 1 . The GAL4 transcription factor binds to binds factor transcription GAL4 The . & Liqun Luo UAS-geneX UAS - cellular autonomous effects (such as as (such effects autonomous cellular - /G ) (where tagged GFP) to signaling molecules molecules GFP) totagged signaling AL reporters arereporters silent. allows This , and can be used in combina in used be can and , 80 80 repressible binary expression system is widely used to manipulate or mark tissues of interest. UAS are in the same animal is is animal same the in are geneX 3 . Thus, when GAL80 is co is .GAL80 Thus, when ) to induce expression of a to) induce expression of 2 UAS-geneX qa is any gene of interest). gene cluster of the the of cluster gene 3 . The combination in in vivo and GAL80) GAL80) and UAS-geneX

expression Drosophila melanogaster . . T hese hese include how to generate and use Q system reagents to express - ­ ­ ­ ­

under conditions of limited glucose levels of seven genes, is required for the catabolism of quinicfilamentous fungusacid (quinate) QS binds to and inhibits QF, and prevents expression of the the of expression prevents QF, and inhibits and to binds QS high, is glucose which in conditions growth Undernormal C/T). sequence the is ‘5 QF for site binding The factor. transcription QF the by induced be the of QF,expression for and sites binding contain seven the for promoters The acid. quinic cluster encode enzymes or cofactors required for the catabolism of hereafter) QF. is a repressor of fiveother The genes in the qa and factor, transcription a is hereafter) and genes: regulatory two tains ( that expression by using the QS inhibitor and MARCM system: expression binary in a subset tissues,of and refinement of GAL4 the as system, applications for same the used be can so and GAL4 the as components basic same the contains system Q The the Applications method of on this treatment with non ( acid quinic containing diet a fed flies in suppressor QS the inhibit can (GAL4, (QF, components of the effector. As such, the Q system contains the same three basic QF the QS suppressor ( factor, a transcription QF the ponents: into introduced system Q The the Development Q of system in for its catabolism. required genes the of expression controls acid) (quinic catabolite for the catabolism quinic of acid as an energy source. In effect, the qa to frominduceexpressionsuppression, from QF QS the allowing QF releases This QS. inhibits and to binds acid quinic present, is acid quinic and limiting is glucose However,cluster.when gene Figs. 2 2 Figs. gene cluster. This results in the expression of the factors required - binding sites and allows for robust QF robust for allows and sites binding qa-1s Figs. 1 UAS and (encoding a protein of 918 aa). qa 918 of protein a (encoding

and GAL80). In addition, the molecule quinic acid acid quinic molecule the addition, In GAL80). and and 3 ). In addition, temporal control of QF activity can can activity QF of control temporal addition, In ).

2 ). Q the system control allows temporal This of QUAS Figs. 1 Neurospora crassa ′ - GGRTAARYRYTTATCC natureprotocols and QS) as the analogous GAL4 system system GAL4 analogous the as QS) and AL and - 4 4 system to generate intersectional toxic molecule ( qa-1f

2 Drosophila ). The (encoding a protein of 816 aa) aa) 816 of protein a (encoding Drosophila 2 . This gene cluster, consisting Department of Biology,of Department Howard QUAS QUAS-geneX

5– | qa VOL.6 NO.8VOL.6 consists of three com three of consists 9 - . This gene cluster con dependent expression expression dependent - Fig. element contains five - genes in in genes 1s (shortened as QS QS as (shortened 1s 1f (shortened as QF QF as (shortened 1f - 3 1 protocol ′ ). ’ is A/G, (R is Y qa effector and and effector | 2011

Neurospora analysis genes can can genes qa |

gene 1105 1,3,1 qa ­ ­ 0

© 2011 Nature America, Inc. All rights reserved. Comparisons with other methods with Comparisons double MARCM’ ( or unlabeled by the GAL4 or Q system. This is called ‘independent mitotic event, then the cell progeny could independently be labeled tion of the QS and GAL80 suppressor were not coupled to the same manipulation of all progeny from a single mitotic event. If segrega mitotic event ( the QS regation of and GAL80 suppressors is coupled to the same the GAL80 repressor). This is called ‘coupled MARCM’, as the seg by system lacks it GAL4 (as the positivelyis sister labeled that cell a and repressor) QS the lacks it (as system Q the by labeled tively posi is that cell one to mitosis by rise give would cell progenitor unlabeled an such, As event. mitotic same the to coupled be can function independently of ety Q system can be used for ( MARCM achieved be analysis can ( manipulations sue tis of precision finer whereby patterns, expression intersectional define to ability the is Highlighted achievable. applications sible GAL4 the with conjunction in used is system binary repressible Q the ( expression QS and and acid quinic using by achieved be expression system has also been used to express effectors effectors express to 1106 used been also has system expression systems. expression Binary protocol b a c

4

| ). However, a key experimental advantage is obtained when when obtained is ).However,advantage experimental key a P2 P2 - QUAS-RFP VOL.6 NO.8VOL.6 in in vivo repressible binary repressiblesystem. binary Promoter-QF QS QS Promoter-QF QUAS-RFP Promoter-Q + + QUAS-RFP applications tubP-QS P1 P1 P1 Fig. 1 QF QF QF | 2011 Fig. 0 F + + + QUAS-RFP ). This allows for the differential marking and ET40-QF QUAS QUAS QUAS 2 |

in in vivo ). natureprotocols 3,10–1 The bacterial bacterial The X X X 3 Quinic acid . As the Q system and GAL4 system × 4 , Q Figure Figure × - MARCM and GAL4 QUAS-RFP ET40-QF tubP-QS Quinic acid Figs. 5 Figs. QUAS QUAS QUAS QUAS QF QF QF 2 QS shows the some pos of Fig. QS – LexA/LexAop X X X X

8 9 ). In addition, the the addition, In ). QUAS-RFP tubP-QS ), which has a vari Quinic acid expression expression + No X X No - MARCM Figs. 1c 1c Figs. binary binary ­ ­ ­ ­ ­ ­

analysis. The The analysis. GAL4 for or patterns expression tional intersec some generate to used be cannot suppressor, hence and GAL80. The LexA/LexAop system does not contain an endogenous sitive to GAL80, whereas the LexA GAL4 activation domain (GAD). The LexA the or VP16 domain activation acidic viral the to fused either is In domain. activation transcriptional a contain the for specific domain (ref. GAL4 of ­independently with permission from reference 4. acid–containing fly food (see Step 6C). Images and schematics are reprinted dashed white circle. The quinic acid–treated flies developed on quinic in promoter. ( a crossing scheme for Q system transgenic flies. tubP indicates the tubulin P1 and P2 indicate promoter 1 and promoter 2. ( ( Figure 1 LexA/LexAop expression binary system for use ers expression compared with of case, this In patterns. expression GAL4 refine to used be could system sion system to drive GAL80 expression. Similarly, the LexA/LexAop Figure resulting GAL4 expression pattern. A better approach, as detailed in the define precisely to difficult it make can suppression.tiveThis determine, and GAL80 levels need to be higher than GAL4 for effec interest of tissue the in GAL80 expressing by achieved be also can patterns patterns. expression Intersectional from a cell division cell a from GAL4 with conjunction in DNA both sister of marking progeny. the for allow also techniques other of number progeny. A both gain independent for used be also can Itevent. mitotic single a fromprogeny all of methods. labeling Mosaic for expression binary or MARCM experiments) used be can that reagent QF new a to contrast (in purposes other GAL4,original and split GAL4 are transgenes not useful for many the as robust as not is GAL4 reconstituted the expression, tional intersec for lines GAL4 characterized existing use cannot GAL4 achieve precise intersectional expression patterns can technique proteins.This GAL4 split the to zippers leucine of reconstituted be can components GAL4 domain activation the containing part other the and DNA the containing part parts—one two into achieved by using the ‘split GAL4’ method, in which GAL4 is split (Step 7B, sion) is not possible. This approach is possible using the Q system limit to GAL4 (using experiment given the lack an of independent repressor LexA,of the reciprocal a ) ) Schematic representing the function of the Q system components. ‘Dual expression control MARCM’ uses uses MARCM’ control expression ‘Dual also is subsets overlapping to patterns expression Limiting b 4 . . Transgenic flies not expressing the RFP reporter are outlined by a . Nonetheless, recent progress has been made to optimize the the optimize to made been has progress recent Nonetheless,. LexA-VP16 - binding domain fused to the GAL4 activation domain) domain) activation GAL4 the to fused domain binding 2

| and Step 7A of the PROCEDURE, is to use a binary expres Schematic and example of Q system components in Fig. 5 c 3,1 ) ) Transgenic 7 . However, GAL80 expression patterns are difficult to LexAop-geneX b would be used to drive ). - and loss and 1 4 . This technique allows labeling of different different of labeling allows technique This . Drosophila LexA operator LexA Coupled MARCM allows the labeling labeling the allows MARCM Coupled - - reporter also has a higher basal level level basal higher a has also reporter of based MARCM to visualize progeny visualize to MARCM based UAS-gen 1 examples of the genotypes shown - 4 function genetic manipulations of of manipulations genetic function ). LexA contains a DNA a contains LexA ). - GAD protein can be inhibited by LexAop-geneX Limiting GAL4 expression expression GAL4 Limiting eX eX or ( LexAop-GAL80 LexAop - b in vivo in independent MARCM MARCM independent - ) ) Diagram illustrating VP16 protein is insen LexA-GAD QUAS-geneX in in vivo . - ), yet it does not not does it yet ), Drosophila reporter expres reporter binding domain domain binding by the addition addition the by 1 8 . However, split Drosophila 15,1 1 (the LexA (the 8 . However, . The two The . 6 - . binding binding report ,LexA

. ­ ­ ­ ­ ­ ­ ­

© 2011 Nature America, Inc. All rights reserved. markers are driven by a ubiquitous promoter, which severely limits system binary of lack the However, a major limitation is low marker expression as a result of after clonal induction,shortly as clones there is examine no perdurance to of a repressorability molecule.the is system binary ible repress a use that methods other over method TSG the of tage double markers (MADM) system for mosaic analysis This is similar in design to the mouse mosaic analysis system with proteins are reconstituted and can be to segregated daughter cells. (FLP/FRT) target in arm trans chromosome same the on teins pro fluorescent chimeric split two places use a binary expression system, but instead a pattern of GAL4 expression pattern a powerful method for resolving the lineage manipulated. Nonetheless, this technique is only, cell progeny cannot be independently GAL4 on based is system the as addition, that lies outside this expression pattern. In which could miss labeling of a cell progeny driver, GAL4 same the by labeled are eny prog both However, transgenes). fewer uses it (as MARCM coupled than design Twin genes. cells, the one each of losing UAS-IR the of loss coupled the follows MARCM the by of loss differential the uses which MARCM, cent proteins. to coupled in design Similar repressorstwo different against fluoresof ( repeat certain neuronal of populations analysis lineage for successfully used has been technique This division. cell a from progeny all of manipulation and labeling prevents this GAL80, by suppressed are tor. However, as both GAL4 driver) that arise from the a common progeni by other the and driver the LexA by labeled (one cells of populations application is listed. applications. The PROCEDURE step describing the (arrows) allow for a number of and combination of these core components inhibitor of QS (quinic acid). The manipulation addition, the Q system includes a small drug the GAL4 and QF suppressors GAL80 and QS. In QF reporters transcription factors GAL4 and QF, the GAL4 and the basic GAL4 and Q system components: the Q system applications. The main box illustrates Figure 2 the utility for tracking complex lineages. As TSG does not use a use not does TSG As lineages. complex tracking for utility the ‘Twin ‘Twin tubP-GAL80 2 2 . On FLPase recombination enzyme/FLPase recognition recognition enzyme/FLPase recombination FLPase On .

repressors | UAS-IR - - α Flowchart of example GAL4 and spot MARCM’ uses UAS uses MARCM’ spot spot generator’ (TSG) does not not does (TSG) generator’ spot - UAS-gen uui pooe) twin promoter), tubulin - spot MARCM is simpler in in simpler is MARCM spot and and ) transgenes as the source source the as transgenes ) eX and 2 - 0 mediated recombination, the two fluorescent fluorescent two the recombination, mediated . This creates two sibling tubP-QS LexA-GAD QUAS-geneX - in vivo based amplification. In addition, both both addition, In amplification. based RNAi repressor (each driven driven (each

14,1 and GAL4 and 9 - . inverse

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X expression X GAL4 NOTQF Combine GAL4

intersection ­ ­ components n UAS mitotic recombination BOX Remove ubiquitous GAL80 expression G80 G4 Coupled MARC Core GAL4components based onsame Regulate GAL8 UAS LexA suppressor. endogenous an require not do designs experimental interest, and of tissue fora reagents havevalidated and generated alreadybeen system LexA if used be could LexA/LexAop system the sufficient, gents rea will grow. useful of Alternatively, availability the system,cases Q in which the the use GAL4 studies system more is notas or effectors recently introduced, a number of Q reagents, such as system. Q the of Limitations expression. transgene by effector lated manipu easily be cannot progeny cell system, binary repressible G4 2 G80 expression X using QF events X Transcription factor GAL4 suppressor GAL4 reporter

Step M 7B 0 QF NOTGAL4 promoter-QF

Regulate QS intersection using GAL4 expression mitotic recombination Remove ubiquitous GAL80 expression based ondifferen

double MARCM Quinic acid QUAS Independent natureprotocols events QS QF lines, remain to be generated. However,generated. be to remain lines, 7C, reporter expression Step Core Qcomponents GAL4 presence X Regulate QF D based on intersection QUAS As the Q system has only been been only has system Q the As QF Steps 1–5 Transcription factor Repressible binary AND t QS suppressor QF suppressor components Combine Q QF reporter X expression X BOX mitotic recombination

Remove ubiquitous QS expression

1 Q-MARCM based on | QUAS VOL.6 NO.8VOL.6 QF QS GAL4 expression GAL4 system with Q-MARCM Regulate QS activity using quinic acid X Also use Step Temporal regulation protocol Full body QF expression of QFactivity 6 QUAS-geneX pattern |

2011

Subset of tissues |

1107 ­ ­

© 2011 Nature America, Inc. All rights reserved. expressed. Differing levels of QS suppression can be achieved by altering the with quinic acid, the developing flies quinic acid or by feeding adult flies quinic acid. If treated in any tissues. This QS-mediated suppression can be inhibited by feeding will result in a progeny that no longer expresses the a by using a tubulin promoter to drive QS ( coupled with quinic acid treatment. Ubiquitous QS expression is achieved Figure 4 into the tion sites can be used to amplify this genomic region for placement A PCR reaction that introduces flanking BamHI and EcoRI restric immediately region upstream of the genomic ATG the start site of a gene be up to the preceding will gene region promoter and involves the cloning of gene first promoters The patterns. expression characterized previously using ics the that recommend be construct used for routine enhancer and promoter cloning therefore We tissues. the potential toxicity of high QF expression in as of because constructs QF generating when desirable or necessary not is level protein increased this cases, most in that, found have Weexpression. protein higher and stability mRNA increased to differ in their 3 recognition sequence and an acontains white that vector transformation a to refers cloning QF constructs: in shown a QF DNA construct. A number of suitable QF DNA constructs are previously characterized enhancer/promoter region of interest into a clone to is approach straightforward most The tissues. target of studies is the generation of Q system reagents for the manipulation Generation of QF transgenic flies. design Experimental with the original expression pattern. longer expressing the the CyO balancer will result in progeny that have a subset of tissues no Crossing this stable expression line with a the To Figure 3 1108 concentration of quinic acid fed to the flies. protocol P1-QF, QUAS-geneX

hr ae he bsc taeis o gnrtn Q transgen QF generating for strategies basic three are There simplify analysis of QS suppression on a QF-induced expression pattern, QUAS-geneX

| VOL.6 NO.8VOL.6

| | pattB-QF-hsp70 Crossing scheme for ubiquitous QS-mediated suppression of QF Crossing scheme for tissue-specific QS suppression of QF. Supplementary TableSupplementary 1 tubP-QS tubP-QS reporter ( ′ P2-QS P2-QS transcriptional terminators. SV40 terminators lead P1-QF, QUAS-mtdT-3xHA | recombinant and selecting against the 2011 QUAS-geneX QUAS-geneX P1-QF, QUAS-mtdT-3xHA QUAS-mtdT-3xHA pattB-QF-hsp70 tubP-QS | ×

construct. natureprotocols +/– quinicacidinfood P1-QF, QUAS-mtdT-3xH Select againstCyO × P2-QS reporter induced by Select againstCyO reporter. This can be directly compared P1-QF, QUAS-mtdT-3xHA

+

genetic marker). These . There are two basic choices for CyO The first step for many Q system tubP-QS ) is recombined with P2-QS (+/– Quinicacidinfood) CyO . In many cases, an enhancer and fly and selecting against ). Crossing pattB-QF-SV40 A QUAS-geneX P1-QF - pattB-QF-hsp70 yet will be tubP-QS CyO attB - unidentified unidentified balancer P1-QF effector

constructs Phi with . (pattB - C31 C31 .

2 4 ­ ­ .

contain an an contain tion for the generation transgenic of animals. The BAC constructs locus regulatory complex a recapitulating of likelihood the increase to promoter-QF-hsp70 artificial chromosome (BAC) recombineeringregion couldassociated with bethe geneused or enhancerto trapinsert insertion. a Bacterial sion pattern of interest, a third strategy is to clone a larger genomic the promoter or the moter would also need to be included, such as either the P trap enhancer an of site insertion the ing mimicked by cloning a large genomic region immediately preced be could trap enhancer an of pattern expression The insertions. trap enhancer with associated region genomic the clone to is est is not expressed in this region. Region 2 expresses both GAL4 is also expressed. Region 1 does not express the final expression pattern reflecting where QF is expressed but not where suppressor ( example, a GAL4 line ( extent of the GAL4 or QF expression pattern. ( intersectional expression patterns. ( Figure 5 as P2-QF expression, which removes the transcription stop cassette, allowing for Region 5 expresses the does not express the cassette can be excised by the activity of the FLPase recombinase. Region 4 cassette ( GAL4 are expressed. The example, region 3 expresses the Q but does not express the b a When the above two approaches fail to recapitulate the expresrecapitulatetheto approaches failabove two When the The second strategy to generate QF expression patterns of inter P2-GAL4 3 2 1 P2 P2 P1 P1 QF-hsp70 G4 G4 G4 QF QF QF -induced expression. Region 6 does not express the 2 7 . These BAC resources are compatible PhiC31with integra

QF NOTGAL4 | QUAS-geneX

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’ indicates P2-GAL4 b 4 QF ANDGAL ) In the ‘QF NOT GAL4’ 4,11,2 5 FRT-transcription stop-FRT c QUAS ) In the ‘QF AND GAL4’ QUAS UA UA G4 QF 6 QF QUAS 5 UA S G4 S induces . In this case, a pro a case, this .In QUAS-geneX S FRT P2-GAL4 FR FLPase UAS-FLPase QUAS>stop>X P2-GAL P1-Q FLPase FR QUAS FR 4 FLPase T T . See Step 7C. T X F STOP

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© 2011 Nature America, Inc. All rights reserved. based on the GAL4 expression pattern. This simplifies the experimentalagainst thesetup balancers will result in progeny that have reducedon theQF expressionthird chromosome. Crossing this stock with any GAL4 lineline recombinedand selecting with a QF NOT intersectional-ready female fly is represented. This QUAS-geneXfly contains a the NOT intersectional strategy to work, four components ( Figure 7 P in shown are DNA constructs trap enhancer QF suitable of number A parallel. in patterns expression new many generate quickly can small a addition, In cloning. by duce repro to difficult is that pattern expression an to rise give often lines. trap enhancer QF of Generation or piggyBac use that P vectors cloning QF and variants coding (C.J.P.,vectors QF attB data). Alternative of unpublished instead P using when improved be its toxicity when broadly expressed (C.J.P.,lished unpublished data). data). Efforts are also underwayto generate,to modify such theas a pan the generation of been codon non reduce sion of QF. This could be due to QF being more toxic thanficult, GAL4. Toespecially for constructs that would result in widespread expres placed at random locations in the genomic insertion of large constructs into intersectional results for many differentthe QF QFlines. expression pattern. This simplifiesthe the balancers experimental will resultsetup forin progenytesting thethaton havethe reducedthird chromosome. GAL4 expression Crossing based this on recombinedstock to anywith QFa line and selecting againstintersectional-ready female fly is represented.and This fly contains a NOT intersectional strategy to work, fourFigure components 6 ( for testing the intersectional results for many different GAL4 lines. - - elements elements are available from the authors on request. element transposase to generate new QF enhancer trap lines. trap enhancer QF new generate to transposase element In addition, success rates for generating QF lines transgenic can In some cases, generating Supplementary Table Supplementary 1 QUAS-GAL80 P1-Q P1-QF

QF

| | P2-GAL4, UAS-mCD8-GFP Crossing scheme for GAL4 NOT QF intersectional experiments. For this Crossing scheme for QF NOT GAL4 intersectional experiments. For P2-GAL4 P2-GAL4 F and expression (and potential QF toxicity), the QF cDNA has P1-QF, QUAS-mCD8-GFP ) need to be combined into a fly. In this example, a GAL4 NOT UAS-QS P1-QF UAS-mCD8-GFP - promoter-QF optimized for P2-GAL4 × CyO andTb Select against QUAS-mCD8-GFP ) need to be combined into a fly. In this example, a × CyO andTb Select against P2-GAL4, UAS-mCD8-GFP - P1-QF, QUAS-mCD8-GF neuronal promoter-QF . These constructs can be injected with with injected be can constructs . These marker, as well as the - constructs that were previously difficult ; lmn o piggyBac or element ; Drosophila QUAS-GAL80 UAS-QS CyO CyO marker as well as the Drosophila synaptobrevin-QF + + - transgenic lines might be dif scale enhancer trap screen screen trap enhancer scale expression. This allows for Enhancer trap lines can can lines trap Enhancer P attP ; P1-QF, P2-GAL4 genome ; QUAS-GAL80 TM6b, Tb UAS-QS sites that have been QUAS-GAL80 TM6b, Tb P1-QF, P2-GAL4 QF UAS-QS - (C.J.P., unpub gene to reduce based vectors vectors based P2-GAL4 28,2 transgene 9 . , transgene UAS-geneX line P1-QF , ­ ­ ­ ­

pattern is determined by the developmental expression pattern of the QF expressionline pattern of the QF line. Conversely, in by the developmental expression pattern of the GAL4 line, and the theyfinal are not equivalent. In strategies limit expression to only regions where GAL4 and QF are expressed,GAL4 lines for their intersection with a characterized QF line. Althoughsimplify both the experimental design required to quickly test many different expression where both QF and GAL4 are expressed. These crossing schemesline and selecting against the balancers will result in progeny that onlycomponents have except for the strategy. These AND intersectional-ready female flies contain all the In necessarythese examples, QF AND intersectional-ready flies are shown for eachwith ( strategies require four components to be combined: There are two strategies to perform an AND intersectional cross. ( Figure 8 and KpnI the ticloningassite the To simplify the generation of additional QUAS-geneX tem reagent is the QF Generation of These reagents are available from the authors on request. tracheal QF addition, no tracheal expression in enhancer traps (C.J.P.,no longer contain the cryptic trachealunpublished enhancer) show decreaseddata). or In the QF coding sequence. Constructs that use QF weak.coding This is likely variants to be due to a cryptic (andweak tracheal enhancer in can show tracheal expression, especially if the trapped enhancers are tissues including neuronal and ( epithelial ( lines has already identified QF enhancer trap lines that label trachea tional QF lines with new expression patterns. A small screen of ~25 posase (e.g., Table 2 authors on request. the from available are vectors piggyBac or terminators hsp70 use that traps enhancer QF terminator. Alternative SV40 an use and P are traps enhancer QF available the that Note and the final expression pattern of the GAL4 line. is is often touseful line on keep the each three of a transgenic single insertions of the same used as the source of the genomic DNA from flies containing the ET14-QF P2-GAL4 P2-GAL4 a QF enhancer traps (and occasionally (and traps enhancer QF y sn P using By ( lines trap enhancer QF existing addition, In pQUAST pQUAST a stop>mCD8-GFP - P1-QF, QUAS> ) XbaI), which allows for easy exchange of inserts between UAS-FLP ) can be mobilized by crossing with a stable P

P2-GAL4 | Crossing scheme for QF AND GAL4 intersectional experiments. tracheal-promoter-QS ), ( glia × - vector ( CyO andTb Select against induced reporter expression (C.J.P., unpublished data). ∆ stop>mCD8-GFP transgenic flies are available ( P1-QF, QUAS> vectors. theIf , - 2–3, Bloomington Stock no. 1798) to generate addi QUAS-geneX QUAS lmn–ae transgenesis element–based CyO ET31-QF

> ; Supplementary Table 1

UAS-FLP stop pUAST - inducible reporter, QUAS-geneX P2-GAL4 a + natureprotocols

> , the resulting expression pattern is determined geneX

mCD8-GFP ; ), imaginal discs ( discs ), imaginal TM6b, effector lines. vector(EcoRI UAS- FLP pUAST-geneX Tb . Crossing these stocks with any GAL4 transgenic lines can be used to inhibit insert or ( OR GAL4 GAL4 construct will be generated. It 4 b P2- P2- stop>mCD8-GFP . b ) P1-QF, UAS> QUAS-geneX QUAS-FLPo UAS-geneX P2-GAL4 QUAS-geneX b promoter-QF Another important Q sys

Supplementary Table , the resulting expression plasmid isnot available, | - ET49-QF × VOL.6 NO.8VOL.6 ) contains the same mul BglII 3 P1-QF CyO andTb Select against 0 stop>mCD8-GFP , many independent independent many , ET40-QF P1-QF, UAS> , - UAS and NotI protocol CyO transgene can be Supplementary Supplementary - - ; transgenic flies,

element > element trans ) ) tissues. QUAS-FLPo

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© 2011 Nature America, Inc. All rights reserved. ing QF transgenicQFing animals,promoter a region expressknownto in QFreporter expression patterns. Similar totheapproaches forclon QS effector lines. of Generation elements. Such be discarded. should lines induction of the minimal a occasionally most and tested effect. for for position lack Eveninducibility of though major chromosomes (X, 2nd, 3rd). Each line transgenic should be Reprinted 20 outlined. are bars, (LH) Scale horn 4. lateral reference and from (MB) calyx permission body with mushroom (AL), lobe antennal The Q-MARCM. by labeled neuron projection olfactory ( 4. reference from modified (*). mutation a for homozygous becomes also It the expressing of capable of copies both lose to progeny cell top the causes here, shown as segregation, chromosome the by followed cross) black (dotted cycle cell the of phase G2 the at recombination mitotic FLP/FRT-mediated mitosis. before or at pacman) (red expression FLPase induces promoter shock FLPase (*). interest of mutation a contain could chromosome sister other The transgene. the of one to Distal (circles). centromere the to distal triangles) same the containing bars) white and bars (black chromosomes sister contains cell parental The cells. of activate to QF for allows which system, FLP/FRT the by mediated event recombination mitotic a by removed is promoter) tubulin the by (driven suppressor QS the of expression ubiquitous ( (Q-MARCM). marker cell repressible a with analysis mosaic 9 Figure 1110 b a protocol tubP-QS ET40-QF P2

CyO G4 P1 | expression is under control of a a of control under is expression VOL.6 NO.8VOL.6 QUAS-geneX QF

hs | Parental cell Coupled MARCM p Schematic and example of Q-based Q-based of example and Schematic , restore QF activity and become become and activity QF restore , FLP QUAS-geneX FRT ; UAS G4 a * tubP-GAL4, 82B G8 tubP ) In a MARCM experiment, experiment, MARCM a In ) ( yw, hsFLP,QUAS-RFP, UAS-GFP tubP QS 0 G8 hsp G QUAS-geneX 0 x QUAS | QF 2011 ). A heat-shock pulse pulse heat-shock A ). FRT QS FRT netos r sln wtot Q inducer, QF a without silent are insertions reporters in a subset subset a in reporters R sites is the the is sites TM6b, Tb insertion (yellow (yellow insertion QUAS-GFP | +

FRT or natureprotocols b hsp70 – , tubP-GAL80 Y d P2 line might be expressed because of of because expressed be might line ) Example crossing strategy for the Q-MARCM experiment ( experiment Q-MARCM the for strategy crossing Example ) Mitotic recombination after DNAreplication G4 P1 QF promoter by local strong enhancer hs marker (G). (G). marker tubP-QS p QS expression can be used to limit

FLP FLP FLP heat- UAS G4 * * G8 QS QS 0 G8 G8

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G

FL 0 0 ET40-QF P

x x × QUAS QF • SelectagainstCyOandTb • Heatshockduringdevelopment QS

+ yw, hsFLP,QUAS-RFP,UAS-GFP yw, hsFLP,QUAS-RFP,UAS-GFP hsFLP µ R m.

; b a , , Two distinctprogeny tubP-GAL4, 82B QF UA UA G4 G4 S (Q-MARCM) Mosaic analysiswithQ-repressiblecellmarker G8 S and and P1 hsp QF 0 G Parental cell G FLP FRT 82B * * G QUAS-geneX QS QS QUAS QUAS QF QUAS FR G8 G8 QF tubP QF QS 0 0 x x T QS QS , tubP-QS R FR R R GH146-QF, 82 T * G , tubP-GAL80 yw, hsFLP,QUAS-GFP ­ TM6b, Tb transgenes can be located on any other chromosome arm. Schematic Schematic arm. chromosome other any on located be can transgenes d c ; the tubulin promoter) have been recombined with FRT sites sites FRT with by recombined been (driven have QS promoter) ubiquitous tubulin the expressing Lines required. is sion vector of choice. NotI KS-QS promoterchoice.of Alternatively, the in promoter tubulin ( definedtissues canclonedbe into a + 82B Supplementary Table1 or For Q For TM6b, Tb Y FR B Mitotic recombination after DNAreplication - FR T aI cud e lnd no n existingpromoter an into cloned be couldEagI) hsp , tubP-QS (that has restriction sites KpnI T QF FLP

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QS QS

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× FL ; P * * •Select againstTb •Heat shockduringdevelopment G yw, hsFLP,QUAS-GF yw, hsFLP,QUAS-GF ptubP-QS-SV40 GH146-QF,82B c 48 h after egg laying. Scale bars, 20 (DAPI). Larvae were heat shocked for 30 min at are labeled with 4,6-diamidino-2-phenylindole a third instar larval wing imaginal disc. Cell nuclei discs. ( second chromosome that expresses QF in imaginal in strategy for the coupled MARCM clone is shown modified from reference 4. ( located on any other chromosome arm. Schematic UAS-geneX homozygous in sister progeny. two independent mutations that can be rendered Figure GAL4 (due to loss of the GAL80 suppressor). See suppressor) and the other that has an active that has an active QF (due to loss of the QS event. This results in two distinct progeny—one an experimentally induced mitotic recombination simultaneously segregated to different progeny by suppressors (driven by the tubulin promoter) are ubiquitous expression of the QS and GAL80 MARCM. ( Figure 10 and and 82B . o eape te EcoRI/KpnI the example, For ). c and QF FR QF d T

Two distinct c , tubP-QS 9 . ( . , progeny d for additional details. ‘*’ and ‘x’ designate d a QUAS .

c ) Example of a coupled MARCM clone in and ) In a coupled MARCM experiment, | QF ET40-QF QUAS , Schematic and example of coupled d QF Box Box ) Example of a single DL1 DL1 single a of Example ) P P QS QS FR QS QS-SV40 - QUAS-geneX ApaI * * G T ol b rpae wt the with replaced be could QS G G 1 is a QF enhancer trap on the ; ), ubiquitous QS expres QS ubiquitous ), codingregionfrom - HindIII transformation vector 82B c d transgenes can be b TM6b, Tb FR ) Example crossing MB Axon T Cell body hsFLP - , tubP-QS EcoRI AL , µ - QF m. containing - LH QS , Dendrite - GAL4 flanked - XbaI pBS- ,

- ­

© 2011 Nature America, Inc. All rights reserved. TA 7B; Step (see patterns expression QF 5 Fig. the limiting with of expression QS direct purpose to used be can patterns GAL4 a using CyO the into inserted ( as balancers TM6B and well as arm chromosome every for Marking different cellular compartments Labeling tissues A Box 1 ? 4. A the Q-MARCMcross willensure thatbothmale and female progeny willcontain Q-MARCMclones. tissue. The Q-MARCM-ready flies often contain an  instar stages. Forimaginal wing discMARCM clones, a30minheat-shock procedure isperformed at48hafteregglaying. For example, togenerate olfactory projection neuron clones, a1.5-hheat-shock procedure canbeperformed from embryonic to third birth date of the tissuesof interest, heat shock the progeny ina37°Cwaterbathfor 30minto2h(see ref. 3. P 82B other chromosome arm.Ifpossible, recombine the which the transgene insertion disruptsproper gene functions. It isrecommended instead toposition the chromosome armwillbecome homozygous afterthe mitotic recombination event, itmight affectthe tissueof interest incases   GH146-QF Q-MARCM–ready flies generated inthe previous step.For example, tobecompatiblewithan 2. Generate a  available for eachmajor chromosome arm( site and control of aheat-shock promoter, (ii)a 1. Generate Q-M performing MARCMexperiments isadapted from identifying the anatomy of asingle neuron. Any QFdriver line canbeusedfor Q-MARCMexperiments ( MARCM experiments canserveavariety of purposes, including generating mosaic tissuesthatare mutant for agene of interest orfor ­GENERATI B

pplication erform Q-M nalyze andexamine Q-M

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CR CR CR CR TROU Analyze Q-MARCMclones using anappropriate technique Cross fivetoten Use standard genetic techniquestocombine aQFline (e.g., Use standard genetic techniquestointroduce the following genetic components into asingle fly:(i) FRT b 1 I I I I ). ), which cansimplifyfuture MARCMexperiments. T T T T UAS-QS | I I I I

CAL CAL CAL CAL tubP-QS Example applications of the Q system. B couldbeused. The LES | promoter-QF

Q-MARCME STEP STEP STEP STEP ARC ARC H

OOT recombined onto the chromosome armof interest ( O transgenic animal ( animal transgenic M cross andgenerate M Thisbalanced stockisavaluable reagent and should bemaintained for future experiments. The developmental time point and extent of the heat shock needs tobeexperimentally determined for eachtarget The Thisbalanced stockisavaluable reagent and should bemaintained for future experiments. M-ready flies N promoter-QF I N Supplementary Table Supplementary promoter-QF O G linethatisQ-M F FLY ST ARC GH146-QF M clones MARCM-ready males with10–20 X insertion canbelocateddistaltothe desired FRT(e.g., PERIMENTS Supplementary Table Supplementary insertion canoccuronany chromosome arm. O QUAS-geneX CKS (1–5GENERATI ARC ARC S upplementary GFP- nuclearLacZ synaptotagmin-HA DenMark (dendrites) EYFP-Golgi EYFP-Mito CD2-HRP mtdT-3xHA mCD8-GFP geneX M clones

M ready 2 ). In addition, by by addition, In ). Nature Protocols α hsFLP -tubu for promoter-QF reporter tovisualize the Q-MARCMclone, such as (mitochondria)

(golgi) QUAS-geneX

insertion onthe Xchromosome (e.g., lin lin (microtubules) (nucleus) ●

T 1 T 0 able 2 ; liveorfixed tissuescanbe used. IMI

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onto the chromosome armoppositetothe used 2 GH146-QF 1 ),

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). N Fig. 9

method of detection or analysis. or detection of method for effectors which determine will question in ment of variety a for Potential applications of the Q system. used. used. G VARIABLE, DEPENDING ) withanFRTchromosome thatusesthe same FRTsiteasthe -ready virgins inafreshly yeastedvial. Depending onthe b O ). Table NS, ~2-10w tubP-QS 1 QUAS-geneX presents a sampling of possible studies, the the studies, possible of sampling a presents

insertions recombined withFRTsitesare in vivo in 82B 82B natureprotocols Fig. 9 applications. In many cases, the experi the cases, many In applications. Immunohistochemistry Live imaging Electron microscopy Immunohistochemistry Live imaging Detection/analysis method FRT FRT , constructs that might be used, the and might that constructs , tubP-QS promoter-QF eeks b ). Using females of these flies for QUAS-mCD8-GFP Fig. FLP recombinase promoter-QF

9 stock,an ) ). The protocol belowfor 10 for additional details). ). However, asthis be can Q The used system FRT

QUAS-geneX | O VOL.6 NO.8VOL.6 (e.g., N

insertion onany , and (iii)anFRT 82B protocol under the FRT promoter-QF, line with | R 3,4,31,32 2011 12,33–35 (continued) effector is is effector eferences

geneX |

1111 ­

© 2011 Nature America, Inc. All rights reserved. 1| P PROCE • • • • • • • • • • EQUIPMENT • • • • • REAGENTS M TA 1112 appropriate Neuronal activation Gene knockdown Report cell activity Cell ablation Ectopically expressing a gene of interest A Mosaic analysis Neuronal inactivation protocol Imaging microscope and software (Zeiss LSM 510 confocal microscope, Zeiss) Chamber 3940, Forma Scientific) Humidified 25°Cincubator to maintainfly crosses (Environmental FLP expression MARCM during experiments) Water at37°Cfor bathset heat GFP filter cube for V8 Penatfluar (KSC295–814DGFPCUBE, Zeiss) RFP filter cube for V8 Pentafluar (KSC295–834DDSRED, Zeiss) Zeiss) Pentafluar, V8 Discovery (Stereo microscope dissecting Fluorescent microscopeDissecting (Stemi 2000, Zeiss) (Wide plugs Fly vial plugs, cat. no. 49–101, http://Flystuff.com) Wide Polystyrene Vials (cat. no. 32–110, http://Flystuff.com) Standard fly Propionic (http://Flystuff.com, acid cat. no. 20–271) 62–103) no. cat. Yeast,Dry Active http://Flystuff.com, Star (Red yeast dry Active (Sigma Quinic acid indiana.edu/Browse/misc Bloomington Stock Center ( Drosophila &cmd=findpub)) addgene.org/pgvec1?identifier=Luo.p9EJQGBAq0qGJ7t4LCsvD2Yax9w vectors ( cloning system Q (many vectors cloning system Q erforming repressible binaryexpression experiments ATER Supplementary Table Supplementary B pplication

LE

In ayeastedvial, cross three tofive | VOL.6 NO.8VOL.6 1 D I ALS URE |

flystocks (many Qsystem fly stocks areavailable from the Example applications of the Q system (continued). - culturing equipment culturing QUAS-geneX | - 2011 Aldrich, cat. no. 138622)

1 - | ) are available from Addgene (http://www. Addgene from available are ) browse/Qintro.htm))

natureprotocols Supplementary TableSupplementary 2 reporter ( - shock (if usingheat shock (if Figs. 1b promoter1-QF TRPA1 Channel Rhodopsin Reporter Reporter (to label clones and/or mutant tissue) tetanus toxin Kir2.1 shibire TRPM8 microRNA against Interfering DNA against GCAMP3 grim hid reaper T Akt Tsc1 geneX tGPH - ; http://flystocks.bio. shock promoter for β and H

(cell growth) (enzyme for synthesis of octopamine) / (PIP3 signaling) Tsc2

(hyperpolarizes neuron) (high temperature activation)

for 2 (low temperature activation) ts1 (neural activity) ; (inhibits vesicle recycling) (cell growth/proliferation)

T able QUAS-geneX (cleaves synaptobrevin) transgenic flies withthree tofive transgenic flies containing the

1

● and geneX (blue light activation)

T IMI S geneX

upplementary 4 °C for ~2 weeks. ~2 for °C 4 at stored be can they but °C), 22–25 at stored if d 3–4 (within fresh used be should Vials overnight. benchtop the on dry to them allow and plugs cotton with vials the Cover covered. are holes all sure making medium, the to tion ~300 Apply sticks. wooden with vials fly standard of Quinic vials acid–containing EQUIPMENT SETUP ~2 in usually sour, smell to begins it when be replaced should container the in paste yeast The use. in not when °C 4 at stored be should paste Yeast consistency. butter peanut creamy achieve to as needed yeast dried additional in Mix dissolved. has paste yeast until spatula metal with Mix acid. propionic (wt/vol) 0.5% with yeast dry active paste Yeast at room temperature (22–25°C). water. 999mlof with isastable This solution stored thatcanbe for months Propionic acid(0.5%(wt/vol)) cycles should be avoided. at vials) acid quinic ten mately the quinic acid. The solution can be stored as 3.5 dissolve help to min ~15 for °C 37 at incubated be to need may solution The tration; saturated concentration is ~300 mg ml Quinic acid solution REAGENT SETUP • • N Three Grade Tweezers 3) Style forcepsSharp (Ted for dissections brain Pella, cat. no. 503, DumontBiology G ~15d (RNAi)

- well glass dissection dishes(Fisher Scientific, dissection well glass cat. no. 21–379)

In a small container, mix approximately equal volumes of of volumes equal approximately mix container, small a In

T able 2

Dissolve quinic acid in water to achieve the desired concen Calcium Calcium imaging Behavior Immunohistochemistry Live imaging Behavior Immunohistochemistry Behavior Immunohistochemistry Two-photon microscopy Immunohistochemistry Behavior Immunohistochemistry Electron microscopy Live imaging Detection/analysis method ).

Poke approximately ten holes into the medium

− In 1

20 °C for months, but repeated freeze/thaw repeated but months, for °C 20 - liter bottle,propionic acid mix5gof

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1 (roughly equivalent to 1.56 M). - ml ml aliquots (makes approxi

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3 weeks. 3

µ l of quinic acid solu acid quinic of l R eferences 3,10,54 50–53 46–49 39–41 36–38 44,45 42,43

­ ­ ­

© 2011 Nature America, Inc. All rights reserved. (B) ( MARCM ( Box D. Ubiquitousexpression of QSthatislinked toamitotic recombination event canalsobeusedfor MARCM(Q-MARCM; of QFduring larvaldevelopment, follow option C.To relieve QSsuppression of QFonlyinadult animals, follow option relieved byquinic acid treatment, resulting intemporal suppression of QF( when performing quinic acid treatment experiments, follow option B. QSsuppression of QF-induced reporters canbe for example, toremove asubsetof QF-labeledtissues, follow option A.To completelyabolishQFexpression, for example, 6| R GAL4 system. repress ortemporally control binary expression ortoStep7carryoutintersectional experiments inconjunction withthe 5| selectable marker). this expression activity can be used to select for recombinant animals (instead of scoring for both copies of the  males. The useof single male crosses ensures thatthe generated stockwillbegenetically homogeneous.  with the progeny togenerate abalanced gene) and setupindividual crosses withvirgin females from anappropriate balancer stock.Carryoutappropriate sib-crosses 4| occurs onlyinfemales and not inmales.  with abalancer stock. T Common expression experiments. Thisrequires thatthe 3| Generating a stable binary expression stock ? expression stockfor subsequent analyses. and progeny atanappropriate developmental stage using anappropriate method (see 2| able A

epression and temporal control of QF-induced binary expression TROU

(ii) ) CR CR CR (i) (i) E QUAS-geneX E 1 QF-induced If desired, usethe balanced stockstoanalyze the effectsof binary expression. Alternatively, proceed toStep6 Select asingle male progeny thatcontains bothcopies of the selectablemarker (usually twocopies of the mini-white It isoften convenient torecombine the Depending onthe goal of the experiment and the identity of I I I

xpressing Q xpressing Q ? ). Coupling bothGAL4-based MARCMand Q-basedMARCMtothe same mitotic event canbeusedfor coupled 2 promoter2-QF insertionsDifferent silenced. of the promoter2-QF,QUAS-geneX crossingthe by thepromoter.same use verified that be should animals This  also beexamined, thatis, inparental flies of genotype reporter willno longer beexpressed evenif QS-mediated suppression of QFusing quinic acid. Inthe former case, inwhich QSisexpressed, the with the genotype T Step 4);maintain onstandard fly food. appropriate food vials. promoter2-QS T T Ubiquitous expression of QScanbeachieved byusing the tubulinpromoter todrive QS( Depending onthe aimof the experiment, either useanappropriate method toanalyze the effectsof QSinF Generate (orselectanexisting)

I I I B ). Choose fivetotenvirgin F TROU

CAL CAL CAL Box LES CR QUAS-geneX I

T H STEP STEP STEP B 2 I OOT CAL LES ). are onthe same chromosome, youmay wishtoproceed directly toStep3generate astablebinary QUAS-geneX If the expression pattern of the S Single males are usedfor establishing balanced recombinant stocksasrecombination does not occurin To get asuccessful recombinant, itisessential touseF S I H tubP-QS

inalltissues inasubsetof tissues N STEP . flies with OOT G reporters are availablewithinsertions oneachof the three major chromosomes ( I

N Promoter2-QS promoter1-QF/QUAS-geneX stock( G expression canbeeffectivelysilenced bythe presence of QS.To refine aQF expression pattern, promoter1-QF/QUAS-geneX recombinant fly to confirm that the that entire confirm to recombinant fly S upplementary 1 females of genotype transgenic lines should express QS in the same pattern as as thepattern same transgenic in express QS should lines promoter2-QS promoter1-QF/QUAS-geneX promoter2-QS promoter1-QF promoter1-QF ●

T T promoter1-QF/QUAS-geneX QF IMI able 2 promoter2-QS line thatresults inthe desired expression patternof QS.Cross ispresent. As acontrol, reporter expression without QSpresence should N G flies (generated inStep4; might need to be tested to find a line that effectively suppresses suppresses effectively that line a find to tested be might to need 2–3 generations ~25–30 d ) and cross with stable and promoter1-QF/QUAS-geneX and promoter1-QF/QUAS-geneX QUAS-geneX QUAS-geneX orraise F geneX stockderived from eachoriginal male. ●

, determine the effectof binary expression onF T IMI 1 heterozygote females asmeiotic recombination reporter onto the same chromosome for future 1 Figs. 1 promoter2-QF toadulthood and proceed toStep6Drelieve reporter can be visualized in live animals, are bothlocatedonthe same chromosome. N G 1 generation ~10–15 d promoter1-QF/QUAS-geneX , T 2 able promoter2-QS Fig. from the progeny inStep1and cross and natureprotocols . 3 1 -reported expression pattern is is expression pattern -reported

) and maintain instandard fly ). Alternatively, if 4 ). To relieve QSsuppression tubP-QS transgenic fly with a transgenic with fly promoter2-QF S

). Selectan | upplementary VOL.6 NO.8VOL.6 QUAS-geneX lines (from promoter1-QF protocol transgenic

| 1 2011

progeny

1

|

1113

+

© 2011 Nature America, Inc. All rights reserved.

( 1114 protocol C Box 2 4. A Using females of these flies for the Q-MARCMcross willensure thatboth male and female progeny willcontain Q-MARCM clones. tissue. The coupledMARCM-ready flies withthe  ­containing 3. P stock. MARCM coupled compatible a generating of simplicity the on depends components promoter2-GAL4 in diagrammed scheme crossing The functions. gene endogenous disrupt homozygous, when lines, these case in used, being site FRT in Q-MARCM for mentioned However, as clones. MARCM coupled   (ii) distally toanFRTchromosome thatusesthe same FRTsiteasthe coupledMARCM-ready flies generated inthe previous step, 2. Generate coupledM used for Q-MARCMexperiments.  ( arm chromosome ( interest of arm chromosome the onto a (iii) a promoter, (ii) heat-shock a of control 1. Generate coupledM both Q-MARCMand GAL4-MARCM techniques. To labelormanipulate allprogeny of amitotic division, coupledMARCMexperiments canbeused( GENERATI

) Quinicacidtreatment of developing flies (ii) (ii) erform coupledM nalyze andexamine coupledM (i)

Figure 10 Figure CR CR CR CR

In a freshly yeasted vial, cross five to ten coupled MARCM-ready males containing Analyze coupledMARCMclones using anappropriate technique Use standard genetic techniquestointroduce the following genetic components into asingle fly:(i) Use standard genetic techniques to introduce the following genetic components into a single fly: (i) (i) fly: single a into components genetic following the introduce to techniques genetic standard Use | promoter2-GAL4 VOL.6 NO.8VOL.6 ? quinic acid solution canbeusedwhen generating quinic acid food vials. rates orthe extent of exposure toquinic acid. To reduce the levelof quinic acid suppression, lowerconcentrations of plates  and larvaeatthe required developmental stages transferred togrape plates orfood containing quinic acid.   tubP-QS promoter1-QF ments. As the outcome of  quinic acid for suppression of QSand re-expression of the approximately every12h toprevent overcrowding of progeny. The developing larval progeny willingest sufficient let them layeggsinquinic acid-containing food vials for 6–12h.Transfer adults tofresh quinic acid food vials at promoter1-QF/QUAS-geneX the quinic acid. Inthe former case, the effectsof ubiquitousQSexpression canbeconfirmed bythe lack of signal from method ( Cross approximately ten Prepare fresh quinic acid–containing food vials (seeREAGENT SETUP). Depending onthe aimof the experiment, either examine the F I I I I

T T T T UAS TROU

I I I I CR CR PAUSE CR CAL CAL CAL CAL QUAS-geneX - I I I tubP-QS | geneX 4 T T T

. However, different tissuesmight respond differently toquinic acid feeding, owing tovariations inproliferation C b STEP STEP STEP B STEP I I I suppression. CAL CAL CAL allows different different allows LES T

and/or and/or | O PO able 2011 O reporter to visualize GAL4 MARCM clones, such as as such clones, MARCM GAL4 visualize to reporter Thisbalanced stockisavaluable reagent and should bemaintained for future experiments. Thisflyline couldalsobe The developmental time point and extent of the heat shock needs tobeexperimentally determined for eachtarget Thisbalanced stockisavaluable reagent and should bemaintained for future experiments. The The UPLED MARCME . Depending on the birth date of the tissues of interest, heat shock the progeny in a 37 °C water bath for 30 min to 2 h.

H S I N and (iii) STEP STEP STEP and ARC upplementary upplementary NT OOT ARC ARC 1 | reporter. As acontrol for effectiveness of O

promoter2-GAL4 Quinic acid fly food can bestored for upto2weeksif kept at4°C. ) orraise F natureprotocols M cross andgenerate coupledM promoter1-QF It ishighly recommended tousea Quinic acid suppression of QSoccurswithin~2hof animals being placedonquinic acid-containing Alternatively, to target aspecific developmental period, crosses couldbesetuponstandard fly food I M-ready fliescontaining M-ready fliescontaining F FLY ST QUAS-geneX N G promoter1-QF promoter-GAL4 tubP-QS ARC canbeexamined. tubP-QS T 1 toadulthood and proceed toStep6Drelieve QS-mediated suppression of QFusing able 2 able could also be combined to other components in the previous step. The positioning of such such of positioning The step. previous the in components other to combined be also could M clones components are present. The lackof either of these components willappearidentical to and and O Fig. 10 Fig. QUAS-geneX animals withapproximately ten CKS (1–6GENERATI ( experiments islackof expression, itisvitaltoknow, with100%certainty, thatboth ). Fig. 10 promoter1-QF or or X b tubP-QS ). ). PERIMENTS promoter-QF tubP-QS b tubP-GAL80 tubP-QS ). reporter to visualize the Q-MARCM clone, such as as such clone, Q-MARCM the visualize to reporter often contain an insertions can technically be located on any chromosome arm to generate generate to arm chromosome any on located be technically can insertions ARC insertions recombined with FRT sites are available for each major major each for available are sites FRT with recombined insertions M clones lines to be used with the same coupled MARCM-ready flies. However,flies. MARCM-ready coupled same the with used be to lines promoter1-QF/QUAS-geneX Box Box 4,1 0 ; liveorfixed tissuescanbe used. UAS-mCD8-GFP ● tubP-QS 1 QUAS-geneX , it is best to avoid recombining these reagents distal to the to distal reagents these recombining avoid to best is it ,

T hsFLP 1 O IMI progeny for suppression of QFusing anappropriate NS, ~2- promoter1-QF/QUAS-geneX , reporter expression of parental flies of genotype insertion onthe Xchromosome (e.g., see N effector( G tubP-GAL80 and (iv) an FRT site and and site FRT an (iv) and VARIABLE, DEPENDING t o 12w recombinant for ubiquitousQSexperi Fig. 1 with 10–20 Fig. 1 c ). QUAS-mtdT-3xHA tubP-GAL80 eeks 0 animals (from Step4)and FLP recombinase FLP ). Thisinvolvescombining Q-MARCM tubP-QS ) -ready virgins recombined recombined recombined , Fig. 10

under the under

O

N b

).

- © 2011 Nature America, Inc. All rights reserved.  (B) QF ( QF driving The final effector expression levelis reflected by whichever transcription factoris driving the final effector transgene (e.g., expression pattern,which couldbemuch broader thanthe expression patternatthe target stage (e.g., the adult stage). they are not equivalent. Whichever line isdriving FLPase expression willcapture the entire developmental profile of that  and QFtransgenes. QF expression patterns. Choose options CorDtolimitexpression of aneffectortoonlytissuesthatexpress bothGAL4 to limitthe extent of GAL4 expression patterns. Choose option BtouseGAL4 expression patterns tolimitthe extent of illustrate the basic principles for performing these genetic experiments. Choose option AtouseQFexpression patterns expression patterns possible, including required genotypes. Beloware details for three of the intersectionals that profile thatisasubset of the GAL4 and QF expression patterns usedinthe experiment. See reference shown in 7| P (D) Quinicacidtreatment of adultflies A erforming intersectional expression experiments (iii) (iii) (iii) (iii) (iv)

(ii) (ii) (ii) ) G CR (i) (i) (i) There are 12intersectional expression patterns possible byusing GAL4 and QFsystems together (examples are I AL ? would no longer express the For example, if  altering GAL4 expression patterns. all four components. The scheme aboveisdesigned tosimplifythe testing of many different    ? described inStep6A. promoter2-QS  ing the T genetic components required ( select for animals containing the balancer chromosome marked bythe S all four genetic components required ( nontoxic toflies and canbesupplemented intheir diet with no adverseeffects. stock ( QUAS-GAL80 is most notable within24h(ref. method ( Recombine in Steps3and 4for solution ( T Cross a To the Analyze Cross a To this Recombine For continued suppression, transfer flies tofresh quinic acid–containing food vials every24–48h.Quinic acid is Analyze adult flies for suppression of QS(as monitored byQF-induced Place adults of genotype Analyze expression atthe appropriate developmental stage using anappropriate technique able 2

upplementary I

NOT TROU TROU

CAL 4 CR CR CR CR CR CR Figs. 2 NOT QUAS I I I I I I G

T T T T T T STEP Fig. promoter1-QF/QUAS-geneX B I I I I I I B ). As acontrol, alsochoose animals that do not contain the promoter2-GAL4/UAS-geneX AL promoter2-GAL4 promoter1-QF Tubby CAL CAL CAL CAL CAL CAL QFintersectional experiments UAS-geneX LES LES T

Fig. > able 4 intersectional experiments , promoter1-QF 5

7 Eventhough the strategies inoptions Cand Dreflect the overlapping intersection betweenQFand GAL4, geneX promoter2-GAL4 transgenes are available oneachchromosome (

H H ). STEP STEP STEP STEP STEP STEP expression canalsoberelieved byquinic acid treatments, asdescribed abovebyusing flies generated as balancer chromosome in OOT and OOT 4 1 promoter1-QF ). T ). Weak suppression of QSisseenwithin6hof being transferred toquinic acid–containing vials, but ). able 2 1 Thisbalanced stockisavaluable reagent and should bekept for future experiments. Thisbalanced stockisavaluable reagent and should bekept for future experiments. Thisbalanced stockisavaluable reagent and should bemaintained for future experiments. These genetic components may belocatedonany chromosome justaslong asthe progeny contains Although quinic acid–mediated relief of ubiquitousQSexpression isdetailed here, tissue-specific I I UAS-geneX N expression using anappropriate technique( N 1 G G promoter1-QF tothe ). Eachof these 12intersectional expression patterns represent aneffectorexpression withthe and the ). As acontrol, alsochoose animals thatdo not contain the tubP-QS and the promoter2-GAL4/UAS-geneX overlapsaportion of the UAS-geneX effectorexpression willbe refined basedonthe expression pattern of the promoter1-QF QUAS-geneX stock, cross in a promoter1-QF/QUAS-geneX

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Make a saturated 300 mg ml Make fresh quinic acid solution Use different QS transgene Use extra copies of QS transgenic lines reporters Use two copies of reporter or marker or GAL4/UAS marker. Try a weaker Verify whether cells are dying by co-labeling cells with antibody Try S shocking for longer time periods (e.g., 1.5–2 h at 37 °C) Try heat shocking at earlier developmental time points. Try heat still present Check that all components (e.g., of UAS-FLPase Perform intersection using different components (e.g., different Use codon-optimized FLPase for higher expression Use extra copy of FLPase or reporter tain all four components Check crossing strategy to ensure that selected progenies con the examined stage. Try the alternative method the developmental readout. The final readout might be weak at by which transcription factor is the final readout and which is There are two approaches for the “AND” intersection. They differ Try a different QF or GAL4 line Use extra copies of the FRT-STOP-FRT reporter Stock no. 30022) Try different QS transgenic line (e.g., olution QUAS-FLPase promoter-QF line or use , , insertion at different genomic loci UAS

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AND natureprotocols acj6-GAL4 projection neurons transgene, GAL4 expression can be used to effectively limit a QF expression pattern. An example of QUAS-geneX GH146-QF is no longer expressed in a subset of dorsal projection neurons because of reporters silent. and . . The

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1 GH146-QF f UAS-QS e QUAS>stop>GFP 1 . A powerful intersectional ). These GH146-QF acj6-GAL4 UAS-FLP QUAS-GAL80 d and AND . . The QUAS-

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. © 2011 Nature America, Inc. All rights reserved. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. com/reprints/index.htm at online available is information permissions and Reprints Published online at interests. CO generated the figures and tables; C.J.P. and L.L. wrote the paper. AUT investigator. University School of Medicine. L.L. is a Howard Hughes Medical Institute by a startup fund from The Center for Sensory Biology at the Johns Hopkins C.-C. Lin and S. Chin for critical reading of the manuscript. C.J.P. is supported A is availableNote: information the via HTML of version Supplementary this article. useful inaddressing cell-cellcommunication orcellnonautonomous effects. could behomozygous for adifferent gene of interest and/or express adifferent effectorgene. Such experiments couldprove clone couldbehomozygous mutant for agene of interest and/or express aneffectorgene. Similarly, the GAL4-marked clone MARCM ( tem and the Qsystemfunction independently, these twomosaic labeling techniquescanbecombined together incoupled suppressor, GAL80, isdifferentially segregated tocellprogeny basedonamitotic recombination event. As the GAL4 sys single olfactory projection neuron isshown in genetically manipulate and labelasmall number of cells, orevenasingle cell.Anexample of aQ-MARCMclone thatlabelsa also bemade homozygous mutants for agene of interest. ThistechniqueiscalledQ-MARCMand isapowerfulapproach to will bereleased from QSsuppression. These cellsthatare positivelylabeled(e.g., marked bya differentially segregate a ckno

M The MARCMtechniquewasoriginally developed for the GAL4 system Ubiquitous QSexpression caneffectivelysilence QF-induced reporter expression. Byusing mitotic recombination to

H PET 14 pruning. axon developmentalregulating in cohesin for function 128 representation.pheromone and fruit segregated spatially centers: (2008). morphogenesis.neuron olfactory regulates Drosophila. in (MARCM) marker crassa. Neurospora in gene qa-1F USA Sci. Acad. expression.gene qa controlinteractinggenes two thatindicates Hered. J. fungi. other and crassa Neurospora in genes acid) (quinic qa the of regulation and Organizationlecture. invitational 1989 Key E. Wilhelmine of cluster gene localizationtheDNA-bindingof domain. identification upstreamof binding sitesgenethe qa clusterin and analysis. mosaic and tracing, lineageexpression, transgene for system binary repressible morphogenesis.neuronal in function gene of Genesis (1993). 401–415 phenotypes.dominantgenerating and fates cell altering Schuldiner,O. G.S.X.E. Jefferis, pathway processing MicroRNA L. Luo, & A.P.,Potter,C.J. Fan, D., Berdnik, cell repressible a with analysis mosaic for protocol A L. Luo, & J.S. Wu, regulatory positive the of regulationAutogenous N.H. Giles, V.B.& Patel, region regulatory qa-1 Neurospora the of analysis Molecular L. Huiet, The M.E. Case, & J. Avalos, D.K., Asch,R.F., Geever, N.H., Giles, R.F.Geever, Baum,J.A.,Geever, Giles, & N.H.R.Expression qa-1Fofactivator protein: a system: Q The L. Luo, & L. E.V.,Liang,Russler, Tasic,B., Potter,C.J., studies for marker cell repressible a with analysis Mosaic L. Luo, & T.Lee, knife. army Swiss geneticist’s fly a Drosophila: in system GAL4 J.B.Duffy, of means a as expressionTargetedgene N. Perrimon, & A.H. Brand, OR w , 227–238 (2008). 227–238 , I

, 1187–1203 (2007). 1187–1203 , N le CONTR

G G FI dgm Fig. 1

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82 NANC , 1–15 (2002). 1–15 , IB ents , 1–7 (1991). 1–7 , et al. et UT

et al. et 0 http://www.natureprotocols.com 81 I Neurospora crassa Neurospora I We thank E. Russler for the image in AL ). Anexample of acoupledMARCMclone inthe wing imaginal discisshown in ONS Cell et al. et DNA sequence, organization and regulation of the qa the of regulation and organizationsequence, DNA , 1174–1178 (1984). 1174–1178 , l I . piggyBac-based mosaic screen identifies a postmitotic a identifies screen mosaic piggyBac-based

NTERESTS

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driven transgene expression in Drosophila with a GAL80 enhancer-trap. GAL80 a with Drosophila in expressiontransgene driven Drosophila. (2010). 16166–16171 combinationDrosophilathewithsystem. Gal4 Drosophila. in systems neuronal connectivity in Drosophila. in connectivity neuronal membranes. pruning. axon developmentalduring degeneratingaxons immunostaining.orimaging live for Press Laboratory Harbor Spring Genetics phiC31. phage from integrase site-specific the using by Drosophila transgenes.expressed precisely engineer to insulator retrovirus gypsy the and effects position Exploiting melanogaster.Drosophila in Drosophila. synapses.neuromuscular Drosophila. in neurons olfactory mice. in markers double with analysis analysis. lineage MARCM. Twin-Spot by revealed as lineageneuronal (2009). 947–953 neurons.of identity and origin developmental the lineage.neuroblast lateral the in architecturesneuronal diverse neurons: lobe antennal expression.transgene of restrictioncombinatorial by functionneuronal of manipulation Genesis Development Drosophila. in differentiation oocyte during network microtubule (2010). 20553–20558 (2004). 678–684 Suster, M.L., Seugnet, L., Bate, M. & Sokolowski, M.B. Refining GAL4- Refining M.B. Sokolowski, & M. Bate, L., Seugnet, M.L., Suster, B.D.Pfeiffer, Yagi,Mayer,R.,Basler, F. Refined& transactivatorsLexA K. in their use and transcriptionalbinary dual with mosaic GeneticT. Lee, & S.-L. Lai, Grieder, N.C., de Cuevas, M. & Spradling, A.C. The fusome organizes the organizes fusome The A.C. Spradling, & M. Cuevas, de Grieder,N.C., L.J. Nicolai, D.R. LaJeunesse, engulf Glia L. Luo, & C. Larsen, J., Perrino,Schuldiner,O., R.J., Watts, dissectingforprotocol A L. Luo, & J.S. Wu, Hawley,R. Scott & W.,Ashburner,M. Sullivan, transgenicof M.P.Construction Calos, & R. Nusse, M., Fish, A.C., Groth, N. Perrimon, & Celniker,S.E. C., Villalta, C., Pitsouli, M., Markstein, K. Venken, B.D.Pfeiffer, L.-H. Ang, E. Fishilevich, Mosaic L. Luo, & Muzumdar,M.D. H.H., Su, J.S., Espinosa, H., Zong, R. Griffin, H.-H. Yu, reveal to MARCM Twin-spotT. Lee, & Y.Huang, L., Shi, C., Chen, H., Yu, Drosophila of analysis ClonalT. Lee, & K. Ito,T., Awasaki, S.-L., Lai, spatial Refined B.H. White, & C.R. Vinson, Peabody,N.C., H., Luan, 3 . Inthiscase, ubiquitousexpression of the GAL4

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